DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells

Abstract

We report the design of a self-assembled aptamer-micelle nanostructure that achieves selective and strong binding of otherwise low-affinity aptamers at physiological conditions. Specific recognition ability is directly built into the nanostructures. The attachment of a lipid tail onto the end of nucleic acid aptamers provides these unique nanostructures with an internalization pathway. Other merits include: extremely low off rate once bound with target cells, rapid recognition ability with enhanced sensitivity, low critical micelle concentration values, and dual-drug delivery pathways. To prove the potential detection/delivery application of this aptamer-micelle in biological living systems, we mimicked a tumor site in the blood stream by immobilizing tumor cells onto the surface of a flow channel device. Flushing the aptamer-micelles through the channel demonstrated their selective recognition ability under flow circulation in human whole-blood sample. The aptamer-micelles show great dynamic specificity in flow channel systems that mimic drug delivery in the blood system. Therefore, our DNA aptamer-micelle assembly has shown high potential for cancer cell recognition and for in vivo drug delivery applications.

Fig. 1.

(A). Schematic illustration of aptamer–micelle formation. All the related sequences are listed in Table 1. (B). Flow cytometric assay to monitor the binding of free TDO5 (250 nM) and with Ramos cells (target cells) and HL60 (control cells) at 37 °C for 5 minutes. The blue and black curves represent the background binding of unselected DNA library or library-micelle. The gray curves represent the binding of TDO5 or TDO5-micelle. (C). Flow cytometric assay to monitor the binding of free aptamer (250 nM) and aptamer–micelle (250 nM based on lipid unit concentration) with target cells (K562) and control cell (CCRF-CEM) at 37 °C for 5 minutes. Neither FITC-KK nor FITC-KB aptamers bind (or bind only weakly) to the target cells. However, FITC-KK-micelle and FITC-KB-micelle show increased binding to the target cells. All probes (FITC-KK, FITC-KB, FITC-KK-micelle, FITC-KB-micelle) do not bind with control cells.

Fig. 2.

Time course of displacement of FITC-TDO5 (A) or FITC-TDO5-micelle (B) bound onto the target cells by competition with an excess of non-labeled TDO5. Cells were incubated with binding buffer containing 250 nM FITC-labeled probes for 20 minutes at 4 °C. Then 2.5 μM non-labeled TDO5 was added to the cells and flow cytometric measurements were carried out at times as shown in the x-axis. The fluorescence intensity before the displacement was normalized to 100% binding. The fluorescence intensity of each data point was normalized to the binding percentage.

Fig. 3.

(A). Design scheme of dye-doped micelles. Bright field and fluorescent images of Ramos cells after incubation with free CellTracker™ Green BODIPY for 2 h (B) and 12 h (C), or incubation with biotin-TDO5-micelle doped with CellTracker™ Green BODIPY for 2 h (D). Image E is the enlarged fluorescent image after postlabeling the biotinylated TDO5 aptamer with QD705 streptavidin. The inset in image C is the enlarged individual cell image. The inset in image E is the fluorescent image of the dead cell. (F). Real-time monitoring of doped special dyes released from the core of the micelles and activated by intracellular enzymes.

Fig. 4.

The enlarged fluorescence image (A), bright field image (B), and stack image after Z-depth scanning (C) of Ramos cells after incubation with TMR-TDO5-micelle in complete cell medium at 37 °C for 2 h. The cross mark in image C indicates that the brightest fluorescence signal comes from inside the cell. (D) Colocalization of TMR-TDO5-micelle (red) and AF633-transferrin (blue) in endosomes.

Fig. 5.

Flow cytometric assay to monitor the binding of 250 nM TDO5-micelle or library-micelle (based on lipid-unit concentration) with cell mixture made by spiking 1 million Ramos (target cells, A,C and D) or HL60 (control cells, B) in 50 uL male whole blood. The incubation time varied from 5 m (A & B) to 1 h (C) and 2 hours (D). The gray and black curves represent the binding of unselected DNA library-micelle and TDO5-micelle, resp.

Fig. 6.

Simplified flow channel response to cell-staining assay. (A). Stepwise immobilization scheme of the flow channel. Representative images of the bright field and fluorescent images of control cells (CCRF-CEM) and target cells (Ramos) captured on the flow channel surface incubated with FITC-TDO5-micelle (B), or FITC-library-micelle (C) or free FITC-TDO5 (D) spiked in human whole blood sample under continuous flow at 300 nL/s at 37 °C for 5 m. All the scale bars are 100 μm.